JP6707028B2 - HIPPO and dystrophin complex signaling in cardiomyogenesis - Google Patents

Description

This application claims priority to US Provisional Application No. 61/913,715, filed December 9, 2013, which is incorporated herein by reference in its entirety.

The present disclosure relates at least to the fields of cell biology, molecular biology and medicine.

Duchenne Muscular Dystrophy Duchenne Muscular Dystrophy (DMD), a fatal hereditary X-linked disorder (Emery, 2002) occurring in 1 in 3500 male births, is characterized by rapid progressive degeneration of skeletal and myocardial fibers. .. Importantly, DMD patients develop heart disease characterized by myocardial necrosis, fibrosis and dilated cardiomyopathy. DMD results from mutations in the dystrophin gene, which encodes a 427 kd cytoskeletal protein present in skeletal, cardiac and smooth muscle cells (Hoffman et al., 1987; Hoffman et al., 1988). In DMD patients, dystrophin expression is abolished, leading to the disruption of the dystrophin-binding glycoprotein complex (DGC), an essential membrane-localized structure in the skeletal and myocardium (Ohlendieck and Campbell, 1991; Ohlendieck et al., 1993). ). A useful mouse model for DMD is the dystrophin-null mdx mouse, which exhibits a disease phenotype similar to human DMD (Bulffield et al., 1984). Although less severe than human DMD, mdx mice have features of human disease such as skeletal muscle degeneration/regeneration and cardiomyopathy after aging.

Introduction to Hippo Signaling The central mammalian Hippo signaling components are the Ste20 kinases Mst1 and Mst2, which are orthologous to the Drosophila Hippo kinase. Mst kinase phosphorylates large tumor suppressor gene homologues (Lats) (Large Tumor Suppressor Homolog) kinase when complexed with Salvador (Salv) scaffold proteins. Mammalian Lats1 and Lats2 are NDR family kinases and are orthologous to Drosophila Warts. Lats kinase then phosphorylates two related transcriptional coactivators, Yap and Taz, which are downstream components of Hippo signaling and partners with transcription factors such as Tead, to regulate gene expression. Yap also regulates gene expression by interacting with β-catenin, an effector of canonical Wnt signaling. Upon phosphorylation, Yap and Taz are removed from the nucleus and become transcriptionally inactive (Fig. 1).

Previous studies of cardiac loss of function in mice have revealed that Hippo signaling inhibits cardiomyocyte proliferation and regulates heart size (Heallen et al., 2011). The Salv deficient heart develops cardiac hypertrophy in which the size of the heart increases by 2.5 times due to cardiomyocyte hyperplasia. In addition, experiments investigating Yap in cardiomyocyte development support the conclusion that Yap is the major Hippo effector molecule during cardiomyocyte development (von Gise et al., 2012; Xin et al., 2011). .. Yap is a cofactor that cooperates with DNA-binding transcriptional regulators. Recent literature indicates that the Tead family cofactor is the major Yap partner (Halder and Johnson, 2011).

The present disclosure relates to methods and compositions that address the longstanding need in the art to provide therapy for cardiac conditions involving at least DMD by targeting the Hippo pathway.

The present invention provides methods and compositions for therapies for at least one medical condition that directly or indirectly affects cardiac muscle cells (cardiomyocytes) in a mammalian individual, including humans, dogs, cats, horses, pigs, etc. Target things. The medical condition can be of any type, including cardiac conditions such as heart failure, cardiomyopathy, myocardial infarction and the like. The medical condition may be a cardiac condition as its initial symptom or cause, or it may be a secondary symptom or cause. The individual may be male or female and may be of any age.

In a particular embodiment, an individual in need of therapy for a cardiac condition is provided with an effective amount of one or more nucleic acids or cells comprising one or more nucleic acids, which nucleic acids provide therapeutic benefit to the individual. To do. In certain embodiments, nucleic acids are in a form that directly or indirectly provides RNA interference, including at least shRNA. In a particular embodiment, the shRNA composition targets a member of the Hippo pathway. The shRNA can target any member of the Hippo pathway, but in certain embodiments it targets Salvador (Sav1).

In embodiments of the present disclosure, there are nucleic acid compositions that target mammalian Sav1, and in certain embodiments, the nucleic acid compositions are shRNA molecules. In certain embodiments, an shRNA comprising one or more of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. There are therapeutic compositions that include the molecule. The composition may or may not be contained in a vector, including viral or non-viral vectors. In a particular embodiment, the shRNA sequence is utilized in a non-integrating vector.

In some embodiments, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, and/or SEQ ID NO:4, Derived nucleic acid containing at least 80% identity to one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. There is an isolated synthetic nucleic acid composition comprising: This derived nucleic acid comprises at least 85% of one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, It may be 90%, 95%, 96%, 97%, 98%, or 99% identical.

In certain embodiments, the nucleic acid comprises the sequence of SEQ ID NO:4 (or SEQ ID NO:5,6,7,8,9,10,11 or 12), SEQ ID NO:4 (or SEQ ID NO:5, 6 respectively). , 7, 8, 9, 10, 11 or 12), and when the sequence and the antisense sequence hybridize with each other to form a double-stranded structure, the sequence and the antisense sequence are , Separated by a loop structure.

In certain embodiments, the nucleic acids are at least 43 nucleotides in length or 137 nucleotides in length or less. In some embodiments, the loop structure is 5-19 nucleotides in length. In a particular embodiment, the derived nucleic acid is compared to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12, respectively. And have 1, 2, 3, 4 or 5 mismatches.

In some embodiments of the composition, the nucleic acid or derived nucleic acid is contained in a vector, such as a viral or non-viral vector. The vector may be a non-integrating vector. The vector may be a non-integrating vector which is a lentivirus vector. In some embodiments of the vector, of the nucleic acids comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. Two or more are on the same vector.

In a particular embodiment, the expression of the nucleic acid is controlled by a cardiomyocyte-specific promoter, such as the rat ventricle-specific cardiac myosin light chain 2 (MLC-2v) promoter; myocardium-specific α-myosin heavy chain (MHC) gene promoter; -137 to +85 minimal heart cell-specific promoters; regulated by tissue-specific or cell-specific promoters such as chicken heart troponin T (cTNT), or a combination thereof.

In certain embodiments, two or more of the nucleic acids comprising SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12. Exist on the same vector. In certain cases, the two or more nucleic acids are regulated by the same regulatory sequence or different regulatory sequences.

In one embodiment, there is a method of treating an individual for a cardiac condition, comprising the step of providing the individual with an effective amount of the compositions encompassed by the present disclosure. In certain embodiments, the individual requires cardiomyogenesis due to the cardiac condition in the individual. In certain embodiments, the individual's heart has cardiomyocyte apoptosis, necrosis and/or autophagy. In certain embodiments, the cardiac condition comprises cardiovascular disease, cardiomyopathy, heart failure, myocardial infarction, ischemia, necrosis, fibrosis or diabetic cardiomyopathy, senile cardiomyopathy. In a particular embodiment, the individual has Duchenne muscular dystrophy. The composition may be given to the individual more than once. The composition may be given to the individual systemically or locally. In certain embodiments, the individual receives additional therapy for the cardiac condition.

In certain embodiments, there is a kit comprising the composition as encompassed by the present disclosure, wherein the composition is held in a suitable container.

In a specific embodiment, there is a method of treating a cardiac condition in an individual, comprising the step of providing to the individual a therapeutically effective amount of a Salvador (Sav1) targeting shRNA. In a particular embodiment, the shRNA is provided to the individual in an AAV9 vector. In certain cases, the individual has Duchenne muscular dystrophy.

In order that the detailed description of the invention that follows may be better understood, the foregoing has outlined, in part, the features and technical advantages of the invention. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It will be appreciated by those skilled in the art that the disclosed concepts and particular embodiments are readily available as a basis for modifying or designing other structures to carry out the same purpose of the invention. It will also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features, which are considered to be characteristic of the invention when considered in conjunction with the accompanying figures, are more apparent from the following description, with regard to both their organization and method of operation, together with further objectives and advantages. It should be well understood. However, it should be explicitly understood that each of the figures are provided by way of illustration and description only and are not intended to define the scope of the invention.

For a more complete understanding of the invention, reference is made herein to the following description in conjunction with the accompanying drawings.

FIG. 7 illustrates an example model of Hippo signaling during development and regeneration; FIG. 8 shows that Yap regulates cell division and motor genes in cardiac regeneration. Heatmap (A) and qRT-PCR validation (B) show transcript levels relative to some Yap targets. *P<0.05, **p<0.001, ***p<0.0001. Exhibit (Morkawa et al., 2014); FIG. 6 shows Salv CKO and Salv CKO;mdx;utrn hearts after ablation. Hearts were excised at the non-regenerating stage 8 days after birth and evaluated by immunohistochemistry 3 weeks after excision. As reported, Salv CKO mutant hearts are capable of regenerating (left panel and (Heallen et al., 2013). In addition, the composite mutant SalvCKO;mdx-; as shown in the right panel. The utrn+/− heart can also be regenerated, in particular the mdx mutant heart cannot regenerate after cardiomyocyte resection; FIG. 6 shows that Hippo ablation promotes recovery of cardiac function after chronic ischemia and established heart failure. Adult hearts were subjected to LAD-O (t=0) and studied 3 weeks later by echocardiography. A failing heart was enrolled in the study and Salv was inactivated by injection with tamoxifen. As indicated, mice were studied by echocardiography at 2-week intervals. Hippo deficient hearts showed functional recovery 2 weeks after tamoxifen injection, with functional recovery completed by 6 weeks; FIG. 9: Effective Salv siRNAs reduce Salv mRNA expression. siRNA were transfected into neonatal cardiomyocytes and Salv mRNA levels were studied by quantitative RT PCR. All three siRNAs effectively reduced Salv mRNA levels; It is a figure which illustrates a mouse Sav1 cDNA sequence. Gray areas indicate examples of shRNA sequences (SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively). Selective exons are indicated by double-underlined versus non-double-underlined sequences. Protein structural domains are indicated by 5'to 3'; by a single underlined sequence in the order WW domain, WW domain, SARAH domain; It is a figure which illustrates a porcine Sav1 cDNA sequence. Gray areas represent examples of shRNA sequences (SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively). Selective exons are indicated by double-underlined versus non-double-underlined sequences. Protein structural domains are indicated by 5'to 3'; by a single underlined sequence in the order WW domain, WW domain, SARAH domain; It is a figure which illustrates a human Sav1 cDNA sequence. Gray areas indicate examples of shRNA sequences (SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 respectively). Selective exons are indicated by double-underlined versus non-double-underlined sequences. Protein structural domains are indicated by 5'to 3'; by a single underlined sequence in the order WW domain, WW domain, SARAH domain;

I. Typical Definitions Unless otherwise specified herein or explicitly denied by context, in the context of the present invention (especially in the context of the following claims) the terms "a" and "an" and "the". And the use of similar referents is intended to cover both singular and plural. The terms "comprising,""having,""including," and "containing" are open-ended terms (ie, including but not limited to, unless otherwise specified). Meaning not). The description of numerical ranges in this specification is intended merely as an abbreviation for individually pointing each numerical value falling within the range, and unless otherwise specified herein, each numerical value is Incorporated in the specification as individually recited herein. Unless specifically stated otherwise herein or explicitly excluded by context, all methods described herein may be performed in any suitable order. The use of any and all examples or example phraseology (eg, “such as”) as used herein is merely intended to better describe the invention and unless otherwise claimed. It does not limit the range. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

As used herein, the term "complementary nucleotide sequence", also known as "antisense sequence", refers to a sequence of nucleic acids that is completely complementary to the sequence of a "sense" nucleic acid encoding a protein. (Eg complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence). Provided herein are nucleic acid molecules comprising sequences complementary to at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

As used herein, the term "corresponding to a nucleotide sequence" refers to the nucleotide sequence of nucleic acids that encode the same sequence. In some examples, when an antisense nucleotide (nucleic acid) or siRNA (inhibitory small RNA) (processed from shRNA) binds to a target sequence, a specific antisense or inhibitory small RNA (siRNA) sequence Is substantially complementary to the target sequence and will therefore specifically bind to the portion of the mRNA encoding the polypeptide. Thus, in general, the sequences of those nucleic acids are highly complementary to the mRNA target sequence and have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base mismatches throughout the sequence. Should be. In many instances, it is desirable to have a perfect match with respect to the sequence of the nucleic acids, that is, a perfect complement to the sequence to which the oligonucleotide specifically binds, and thus a zero mismatch along the complementary region. is there. Highly complementary sequences will generally bind very specifically to the target sequence region of the mRNA, and thus highly efficiently reduce and/or even inhibit translation of the target mRNA sequence into a polypeptide product. Become. See, eg, US Pat. No. 7,416,849.

Substantially, the complementary oligonucleotide sequence is more than about 80 percent complementary (or "% perfect match") to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, more preferably the oligonucleotide is It is more than about 85 percent complementary to the corresponding mRNA target sequence that specifically binds. In certain embodiments, as described above, it is desirable to have oligonucleotide sequences that are even more complementary when used in the practice of the invention, in such instances the oligonucleotide sequences are those to which the oligonucleotide specifically binds. Greater than about 90 percent complementary to the corresponding corresponding mRNA target sequence, and in certain embodiments greater than about 95 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, The designed oligonucleotide may be complementary to the target mRNA to which it specifically binds by up to 96%, 97%, 98%, 99%, or even 100% perfect match. See, eg, US Pat. No. 7,416,849. The percent similarity or percent complementarity of any nucleic acid sequence can be determined, for example, by utilizing any computer program known to those of skill in the art.

As used herein, the term "knockdown" or "knockdown technology" refers to gene silencing technology in which the expression of a target gene or gene of interest is reduced compared to gene expression prior to introduction of shRNA. , That technique can result in inhibition of production of the target gene product. The term "reduced" is used herein to indicate that target gene expression is reduced by 0.1-100%. For example, expression of 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or even 99. % Can be reduced. Expression is expressed, for example, in 2 to 4, 11 to 14, 16 to 19, 21 to 24, 26 to 29, 31 to 34, 36 to 39, 41 to 44, 46 to 49, 51 to 54, 56 to 59, 61. ~64, 66-69, 71-74, 76-79, 81-84, 86-89, 91-94, 96, 97, 98 or 99 etc. can be reduced to any amount (%). .. Knockdown of gene expression can be guided by the use of siRNA or shRNA.

As used herein, the term "nucleotide sequence" refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, and may include synthetic, non-natural or modified nucleotide bases that can be incorporated into the DNA or RNA polymer. Optionally contained. The term "polynucleotide" is used interchangeably with the term "oligonucleotide". Unless explicitly stated, the term "nucleotide sequence" is interchangeable with "nucleic acid sequence". "Nucleotide sequence" and "nucleic acid sequence" are terms that refer to a sequence of nucleotides in a polynucleotide molecule.

As used herein, the term “operably linked” refers to nucleic acid sequences being linked together on a polynucleotide such that the function of one of the sequences is affected by the other. For example, if the two sequences are located so that the regulatory DNA sequence affects the expression of the coding DNA sequence (ie, the coding sequence or functional RNA is under the transcriptional control of a promoter), the regulatory DNA sequence is RNA ("RNA coding sequence" or "shRNA coding sequence") or a DNA sequence encoding a polypeptide is said to be "operably linked." The coding sequence can be operably linked to regulatory sequences in sense or antisense orientation. An RNA coding sequence refers to a nucleic acid that can function as a synthetic template for RNA molecules such as siRNA and shRNA. Preferably the RNA coding region is a DNA sequence.

As used herein, the term "pharmaceutically acceptable" refers to carriers, diluents, excipients and/or salts that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, suspension or emulsion or as described elsewhere in this specification.

The term "promoter", as used herein, is usually located upstream (5') to the coding sequence and allows for recognition by the RNA polymerase and other factors necessary for proper transcription. Refers to nucleotide sequences that direct and/or control expression. A "promoter" is a short DNA sequence composed of a TATA box, which contains the minimal promoter and other sequences that serve to specify the site of transcription initiation and to which regulatory elements are added to control expression. "Promoter" also refers to a nucleotide sequence containing a minimal promoter plus regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Thus, an "enhancer" is a DNA sequence that stimulates promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. It is capable of acting in both directions (sense or antisense) and is functional when moved either upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA binding proteins that mediate their effects. A promoter may be obtained in its entirety from a natural gene, may be composed of different elements derived from different promoters found in nature, or may be composed of synthetic DNA segments. Promoters can also contain DNA sequences involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. Any promoter known to those of skill in the art to regulate expression of shRNA or RNA coding sequences is contemplated in the practice of the invention.

As used herein, the term "reporter element" or "marker" means a polynucleotide that encodes a polypeptide that can be detected in a screening assay. Examples of polypeptides encoded by reporter elements include, but are not limited to, lacZ, GFP, luciferase and chloramphenicol acetyltransferase. See, eg, US Pat. No. 7,416,849. Many reporter elements and marker genes are known to those of skill in the art and are envisioned for use in the inventions disclosed herein.

As used herein, the term "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. "Messenger RNA transcript (mRNA)" refers to RNA that is free of introns and that can be translated into protein by the cell.

As used herein, the term "small interfering" or "short interfering RNA" or "siRNA" refers to a nucleotide RNA that is capable of inhibiting the expression of a gene sharing a homology that targets a desired gene. Refers to double strands. The RNA duplex forms 15,16,17,18,19,20,21,22,23,24 or 25 base pairs and carries a 2 nucleotide 3'overhang, 15,16,17,18. , Two complementary single-stranded RNAs of 19, 20, 21, 22, 23, 24 or 25 nucleotides. RNA duplexes are formed by complementary pairing between two regions of an RNA molecule. The siRNA is "targeted" to the gene, where the nucleotide sequence of the double-stranded portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In some embodiments, the duplex length of the siRNAs is less than 30 nucleotides. The duplex is 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. Good. The duplex length may be 17-25 nucleotides in length. Double-stranded RNA can be expressed in cells from a single construct.

As used herein, the term "shRNA" (small hairpin RNA) refers to an RNA duplex where a portion of the siRNA is part of the hairpin structure (shRNA). In addition to the double-stranded portion, the hairpin structure can contain a loop portion located between the two sequences forming the double strand. Loops can vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3'or 5'overhangs. In some embodiments, the overhang is a 3'or 5'overhang of length 0, 1, 2, 3, 4 or 5 nucleotides. In one aspect of the invention, the nucleotide sequence in the vector functions as a template for expressing a small hairpin RNA containing a sense region, a loop region and an antisense region. After expression, the sense and antisense regions form a duplex. It is this duplex that forms the shRNA, which hybridizes to, for example, Sav1 mRNA and reduces Sav1 expression.

As used herein, the term "treating" improves at least one symptom of a disease or disorder such as, but not limited to, ischemic heart disease, heart failure, cardiomyopathy, etc. To cure and/or prevent.

As used herein, the term "vector" may or may not be self-propagating or mobile, and may be present by integration into the cell genome or extrachromosomally. Any viral or non-viral vector capable of transforming a prokaryotic or eukaryotic host cell with any of (for example, an autonomously replicating plasmid having an origin of replication), and a double-stranded or single-stranded linear or Refers to any plasmid, cosmid, phage or binary vector that is circular. Any vector known to those of skill in the art is contemplated for use in the practice of this invention.

II. General Embodiments Embodiments of the present disclosure relate to methods and compositions for treatment of cardiac pathologies, including those in which cardiomyocytes need to be renewed. Cardiomyocytes may need to be renewed for any reason including, for example, disease, underlying genetic status, and/or trauma. In certain embodiments, the individual has cardiac fibrosis, such as cardiomyocyte damage, necrosis, and/or Duchenne muscular dystrophy (DMD).

In certain embodiments, the methods and compositions are applied to individuals with DMD. The diseased heart in DMD patients has extensive cardiomyocyte damage, necrosis and fibrosis. Similarly, mdx mutant mice modeling human DMD have heart failure and cardiomyopathy with severe fibrosis and dilation. The Hippo signaling pathway was identified as a key suppressor of cardiac regeneration after tissue cutting or myocardial infarction (Heallen et al., 2013). In addition, the data show that Hippo regulates transcription of DMD-related genes in the heart. Taken together, Hippo signaling was thought to inhibit the repair response to Duchenne cardiomyopathy. As shown herein, in contrast to regenerative wild-type neonatal hearts, neonatal mdx hearts are unable to regenerate after lumpectomy. Most notably, this regenerative capacity was largely restored in the Hippo;mdx composite mutant heart, indicating gene repression of the mdx heart phenotype. Taken together, modulating Hippo signaling functions as a powerful tool for repairing heart muscle, such as DMD heart muscle.

In addition to ameliorating DMD cardiomyopathy, inhibition of the Hippo pathway after established heart failure (HF) resulted in a significant improvement in cardiac function in a mouse model of ischemic cardiomyopathy and HF. Be done. Mice with established ischemic cardiomyopathy and HF to deplete the Hippo pathway were generated by waiting 3 weeks after induction of myocardial infarction. At 3 weeks, the Hippo pathway was inactivated and cardiac function was followed at 2 week intervals by echocardiography. Hippo deficiency strongly enhances cardiac repair in established HF, whereby the Hippo mutant restores function comparable to the non-operated sham control.

Presented herein are a set of three unique but typical short hairpin RNAs (shRNAs) that specifically target the Hippo pathway member Salvador (Sav1). shRNA results in a selective reduction of Sav1 mRNA levels, similar to gene knockout in a mouse model. In certain embodiments, shRNA can be delivered using an AAV9 (adeno-associated virus serotype 9) vector with tropism for the heart. In particular embodiments of the present disclosure, shRNA sequences of nucleotides specific to the target Sav1 are envisioned.

III. Salvador
In a specific embodiment, the Hippo pathway member Salvador (salvador family WW domain-containing protein 1) is targeted by shRNA in the treatment of cardiac pathologies. The gene may be referred to as salvador homolog 1, Salv, SAV1, SAV, WW45 or WWP4. A representative nucleic acid is provided under GenBank® Accession No. CR457297.1 and a representative protein sequence is provided under GenBank® Accession No. Q9H4B6.

The gene encodes a protein containing two WW domains (a modular protein domain that mediates specific interactions with protein ligands) and a coiled-coil region. It is ubiquitously expressed in adult tissues. It also contains a SARAH (Sav/Rassf/Hpo) domain at the C-terminus (three eukaryotic tumor suppressor genes that give the domain its name). In the Sav (Salvador) and Hpo (Hippo) families, the SARAH domain mediates signal transduction from Hpo through the Sav scaffold protein to the downstream component Wts (Warts); phosphorylation of Wts by Hpo leads to cyclin E, Diap1. And down-regulate other targets to trigger cell cycle arrest and apoptosis. The SARAH domain may also be involved in dimerization.

IV. Exemplary Method of Treatment In an embodiment of the present disclosure, there is a method of treating a cardiac condition in an individual using a nucleic acid that targets the Sav1 gene. In certain embodiments, the cardiac condition comprises cardiomyocytes that need to be renewed, for example, either because of disease (eg, affected or genetic) or trauma. In certain embodiments, there is a heart that is diseased in the individual. An individual can have cardiomyocytes that need to be neoplastic for any reason. For example, cardiomyocytes in an individual may be apoptotic, autophagic, or the tissue may be necrotic.

In certain embodiments, the individual has heart failure, cardiac fibrosis, cardiomyopathy, ischemic cardiomyopathy, myocardial necrosis, dilated cardiomyopathy, skeletal and/or myocardial fiber degeneration, diabetic cardiomyopathy, senile cardiomyopathy, etc. Can have. In certain embodiments, the methods of the present disclosure allow the ability of cardiomyocytes to re-enter the cell cycle. An individual may need to improve cardiac function for any reason, including due to age, disease, trauma, etc.

In a particular embodiment, the individual is provided with an effective amount of a Sav1-targeting nucleic acid so that cardiomyocytes present in the individual can be renewed. In another embodiment, a nucleic acid that targets Sav1 is provided to the individual, eg, the nucleic acid is already present at the time of delivery in cells, including cardiomyocytes or stem cells.

The nucleic acid composition of the present disclosure can be given to an individual once or more than once. Delivery may occur when cardiomyogenesis is diagnosed or a cardiac condition is diagnosed. Delivery can be to individuals susceptible to cardiac conditions, such as individuals or family history, overweight, high cholesterol, and/or smokers. Delivery may be discontinued or continued once it is determined that the cardiac symptoms have improved and/or cardiomyocytes have been renewed.

V. Nucleic Acids Targeting Sav1 In particular embodiments, there is one or more nucleic acids targeting Sav1 such that the expression of Sav1 is detectably reduced. The nucleic acid may be DNA or RNA, but in certain embodiments the nucleic acid is RNA such as shRNA.

In one embodiment, the shRNA is a "hairpin" or stem-loop RNA molecule that comprises a sense region, a loop region and an antisense region complementary to the sense region. In other embodiments, the shRNA comprises two different RNA molecules that bind non-covalently to form a duplex. See, eg, US Pat. No. 7,195,916.

In certain cases, shRNA is a single-stranded RNA molecule that forms a stem-loop structure in vivo and may be about 40-135 nucleotides in length. In at least certain cases, the 5-19 nucleotide loop links two complementary 19-29 nucleotide long RNA fragments that form a double-stranded stem by base pairing. Transcription and synthesis of shRNAs in vivo is driven by the Pol III promoter, and the resulting shRNAs are then cleaved by the RNase III enzyme, Dicer, to produce mature siRNAs. The mature siRNA enters the RISC complex. Thus, in certain embodiments, shRNAs for the inhibition of Sav1 expression according to the present disclosure contain both sense and antisense nucleotide sequences.

The present disclosure provides specific examples of Sav1-targeting shRNAs (SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, or 12), although other shRNA compositions can be utilized. .. One of ordinary skill in the art can identify suitable sequences in any manner, but in certain embodiments, genes from two or more organisms are aligned to search for overlapping regions for amino acid coding sequences to determine specific lengths. Sequencing for regions (eg, 19 nt), for sequences with repeats of 3 nt or less, and/or BLAST potential sequences to <15 bp relative to any other portion of the human genome. You can ensure that there is only homology.

When properly targeted to a particular mRNA in cells by its nucleotide sequence, shRNA specifically represses Sav1 gene expression. In at least some cases, shRNAs can reduce the cellular levels of specific mRNAs and the levels of proteins encoded by such mRNAs. shRNAs are sequence-specific by degrading by targeting mRNAs utilizing sequence complementarity. Thus, they can be highly target-specific and have been shown to target mRNAs in mammals that are encoded by different alleles of the same gene.

In certain embodiments, shRNAs targeting regions of the target gene that are to be downregulated or knocked down are expressed in cells. The shRNA duplex may be substantially identical in sequence (eg, at least about 80%, 81%, 82%, 83%, 84%) to the sequence of the target gene for downregulation. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical). In certain embodiments, there are no more than 5 mismatches between the shRNA sequence and the target Sav1 sequence. In certain embodiments, a minimum of 18 bp of homology is utilized in the region of complementarity between the shRNA sequence and its target. In a specific embodiment, a specific assay is utilized to test the appropriate mismatch for the shRNA and its target. In certain embodiments, algorithms can be utilized to identify appropriate mismatches for shRNA and its target.

Therefore, note that perfect complementarity between the target sequence and the shRNA is not required. That is, the resulting antisense siRNA (after shRNA processing) is sufficiently complementary to the target sequence. The sense strand is substantially complementary to the antisense strand in order to anneal (hybridize) to the antisense strand under biological conditions.

In particular, the complementary polynucleotide sequence of the shRNA can be designed to specifically hybridize to a particular region of the desired target protein or mRNA to interfere with replication, transcription or translation. The term “hybridize” or variations thereof refers to a sufficient degree of complementarity or pairing between the antisense nucleotide sequence and the target DNA or mRNA such that stable and specific binding occurs therebetween. In particular, 100% complementarity or pairing is desirable, but not required. The antisense nucleotide sequence is defined under defined conditions, eg, under physiological conditions, preferably for in vivo therapy, and substantially without nonspecific binding of the antisense nucleotide sequence to the non-target sequence. If sufficient hybridization occurs between the target nucleic acid of interest and the target nucleic acid of interest, specific hybridization has occurred. Preferably, specific hybridization interferes with the normal expression of the gene product encoded by the target DNA or mRNA.

For example, the antisense nucleotide sequence is designed to specifically hybridize with the replication or transcriptional regulatory region of the target gene, or the translational regulatory region such as the translation initiation region and exon/intron junction, or the coding region of the target mRNA. be able to. In certain embodiments, the shRNA targets a sequence encoding the N-terminal region of the Sav1 protein, the sequence encoding the center of the Sav1 protein, or the sequence encoding the C-terminal region of the Sav1 protein.

shRNA: Synthesis As is generally known to those skilled in the art, commonly used oligonucleotides are combinations of naturally occurring purine and pyrimidine bases, sugars, and covalent internucleosides that include a phosphate group in a phosphodiester bond. A ribonucleic acid or deoxyribonucleic acid oligomer or polymer having a bond. However, it should be noted that, as described below, the term “oligonucleotide” also encompasses various non-naturally occurring mimetics and derivatives of naturally occurring oligonucleotides, ie modified forms.

The shRNA molecules of the present disclosure can be prepared by any method known to those of skill in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known to those skilled in the art, such as solid-phase phosphoramidite chemical synthesis. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding shRNA molecules. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize shRNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

shRNA molecules can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA/RNA synthesizers. Custom shRNA synthesis services are available from commercial suppliers such as Ambion (Austin, Tex., USA) and Dharmacon Research (Lafayette, Colo., USA). See, eg, US Pat. No. 7,410,944.

Various well-known modifications can be introduced to DNA molecules as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo or deoxynucleotides to the 5'and/or 3'ends of the molecule, or phosphorothioate or 2'O rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. -There is the use of methyl. The antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known to those of skill in the art. Antisense oligonucleotides are naturally occurring nucleotides, or a variety of nucleotides designed to enhance the biological stability of a molecule or the duplex formed between an antisense and a sense nucleic acid. Can be chemically synthesized using various modified nucleotides (for example, phosphorothioate derivatives and acridine-substituted nucleotides can be used).

The shRNA molecule of the present invention may be various modified equivalents of the structure of any Sav1 shRNA. By "modified equivalent" is meant a modified form of a particular siRNA molecule having the same target specificity (ie, recognizing the same mRNA molecule that complements an unmodified particular siRNA molecule). Thus, a modified equivalent of an unmodified siRNA molecule is a modified ribonucleotide, ie, a ribonucleotide containing a modification in the chemical structure of the base, sugar and/or phosphate (or phosphodiester bond) of the unmodified nucleotide. Can have. See, eg, US Pat. No. 7,410,944.

Preferably, the modified shRNA molecule has a modified backbone or a non-natural internucleoside linkage, such as a modified phosphorus-containing backbone and a morpholino backbone; siloxanes, sulfides, sulfoxides, sulfones, sulfonic acids, sulfonamides, and sulfamic acid backbones; formacetyl and It contains non-phosphorus skeletons such as thioform acetyl skeletons; alkene-containing skeletons; methyleneimino and methylenehydrazino skeletons; See, eg, US Pat. No. 7,410,944.

Examples of modified phosphorus-containing skeletons include, but are not limited to, phosphorothioates, phosphorodithioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramites. There are darts, thionophosphoramidates, thionoalkylphosphotriesters and boranophosphates and their various salt forms. See, eg, US Pat. No. 7,410,944.

Examples of non-phosphorus-containing scaffolds described above are known to those of skill in the art, eg, US Pat. No. 5,677,439, each of which is incorporated herein by reference. See, eg, US Pat. No. 7,410,944.

Modified forms of shRNA compounds include modified nucleosides (nucleoside analogs), ie modified purine or pyrimidine bases such as 5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-ones, pyridin-2-ones, phenyls, pseudouracils. , 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (for example, 5-methylcytidine), 5-alkyluridine (for example, ribothymidine), 5-halouridine (Eg, 5-bromouridine), or 6-azapyrimidine, or 6-alkylpyrimidine (eg, 6-methyluridine), 2-thiouridine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethyl. Aminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2 -Thiouridine, 4-acetylcytidine, 3-methylcytidine, propyne, keusin, wibutosin, wibutoxin, β-D-galactosylkeusin, N-2,N-6 and O-substituted purines, inosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 2-methylthio-N-6-isopentenyladenosine, β-D-mannosylke It may also contain wocin, uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivative and the like. See, eg, US Pat. No. 7,410,944.

In addition, modified shRNA compounds can also have substituted or modified sugar moieties, such as 2'-O-methoxyethyl sugar moieties. See, eg, US Pat. No. 7,410,944.

In addition, to assist in the design of shRNAs to effectively silence any target gene, some vendors “select” an shRNA given the target gene's mRNA or coding DNA sequence. We maintain a web-based design tool that utilizes these general guidelines for: Examples of such tools are found in Dharmacon, Inc. (Lafayette, Colo.), Ambion, Inc. (Austin, Tex.) website. For example, the selection of shRNAs involves choosing a site/sequence unique to the target gene (ie, a sequence that does not share significant homology with genes other than the target gene), so that other genes are identified by this identity. Will not be targeted by the same shRNA designed for the target sequence of

Another criterion to consider is whether the target sequence contains a known polymorphic site. When a polymorphic site is included, a single base mismatch between the target sequence and the complementary strand in a given shRNA can significantly reduce the effectiveness of RNAi induced by that shRNA, thus targeting certain alleles. The shRNA designed to do not effectively target another allele. Given the target sequences and such design tools and design criteria, one of ordinary skill in the art in possession of the present disclosure should be able to design and synthesize additional siRNA compounds useful in reducing Sav1 mRNA levels. Is.

shRNA: Administration The present disclosure provides compositions of polymers or excipients and one or more vectors encoding one or more shRNA molecules. The vector can be formulated into a pharmaceutical composition with a suitable carrier and administered to a mammal using any suitable route of administration.

Due to this accuracy, the side effects commonly associated with conventional drugs can be reduced or eliminated. In addition, shRNAs are relatively stable and they can be modified like antisense to improve pharmaceutical characteristics such as increased stability, deliverability and ease of manufacture. In addition, shRNA molecules utilize the natural cellular pathway, RNA interference, and are therefore highly efficient in degrading targeted mRNA molecules. As a result, achieving therapeutically effective concentrations of shRNA compounds in a subject is relatively easy. See, eg, US Pat. No. 7,410,944.

The shRNA compound can be administered to the mammal by a variety of methods via different routes. They can also be delivered directly to a particular organ or tissue by any suitable localized administration method, such as direct injection into the target tissue. Alternatively, they can be delivered encapsulated within liposomes by iontophoresis or by incorporation into other media such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

In vivo inhibition of specific gene expression by intravenously injected RNAi has been achieved in various organisms including mammals. In a particular embodiment, the shRNA molecule is contained in a vector, including viral or non-viral vectors. In certain embodiments, the vector is not integrated, but in other embodiments it is. Viral vectors can be, for example, lentivirus, adenovirus, adeno-associated virus, and retrovirus. Non-viral vectors include plasmids. In a particular embodiment, the AAV9 vector (Piras et al. 2013) is utilized. The vector may be delivered to the individual systemically or locally. In certain embodiments, the vector utilizes a cell-specific promoter, such as a tissue-specific or cardiomyocyte-specific promoter. In certain embodiments, the vector is delivered by local injection.

One of the routes of administration of the shRNA molecules of the invention is direct injection of the vector at the desired tissue site, such as into diseased or ischemic heart tissue.

In general, a polynucleotide sequence and a single segment encoding a first segment, a second segment immediately 3'to the first segment, and a third segment immediately 3'to the second segment. Included in the invention is a vector comprising a promoter operably linked to an isolated nucleic acid sequence, wherein the first and third segments are each shorter than 30 base pairs in length and longer than 10 base pairs in length, respectively. The sequence of the third segment is the complement of the sequence of the first segment. The second segment, located immediately 3'to the first segment, encodes a loop structure containing 4-10 (ie, 4, 5, 6, 7, 8, 9, 10) nucleotides. The nucleic acid sequence is expressed as a siRNA and functions as a small hairpin RNA molecule (shRNA) that targets the designated nucleic acid sequence.

More specifically, the invention includes compositions and methods that selectively reduce the expression of Sav1-derived gene products. The invention provides a vector comprising a polynucleotide sequence comprising a nucleic acid sequence encoding a Sav1-targeting shRNA. shRNA forms a hairpin structure including a double-stranded structure and a loop structure. The loop structure can contain from 4 to 10 nucleotides, such as 4, 5 or 6 nucleotides. The duplex is less than 30 nucleotides in length, such as 10-27 nucleotides. The shRNA can further include an overhang region. Such protrusions may be 3'overhanging regions or 5'overhanging regions. The overhang region may be, for example, 1, 2, 3, 4, 5, or 6 nucleotides in length.

The invention provides, inter alia, a method of treating a mammal by administering to the mammal a composition comprising one or more of the vectors described herein. In one aspect of the invention, multiple vectors, each encoding a different shRNA (which targets a different region of the Sav1 nucleic acid sequence), can be administered to the mammal simultaneously or sequentially. Each vector can encode multiple shRNAs targeting different regions of the same gene; that is, 2 of the shRNA comprising SEQ ID NO: 10 and the shRNA comprising SEQ ID NO: 11 and the shRNA comprising SEQ ID NO: 12. ShRNAs containing more than one can be encoded. In another embodiment, each vector encodes multiple copies of shRNA comprising SEQ ID NO: 10 or multiple copies of shRNA comprising SEQ ID NO: 11 or multiple copies of shRNA comprising SEQ ID NO: 12 in any ratio. You can

The vector of the present invention may further contain a promoter. Examples of promoters include regulatable and constitutive promoters. For example, the promoter may be the CMV or RSV promoter. The vector may further include a polyadenylation signal such as a synthetic minimal polyadenylation signal. Many such promoters are known to those of skill in the art and are envisioned for use in this invention. In other examples, the promoter may be a tissue-specific promoter, such as a cardiac tissue-specific promoter.

The vector can further include one or more marker genes or reporter genes. Many marker genes and reporter genes are known to those of skill in the art. The present invention contemplates the use of one or more marker genes and/or reporter genes known to those of skill in the art in the practice of the present invention. Marker genes or reporter genes provide a method for tracking the expression of one or more linked genes. Marker genes or reporter genes for intracellular expression provide products, usually proteins, detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. Gene expression products, marker genes or reporter genes, regardless of whether they are derived from the gene of interest, can also be detected by labeling. Labels contemplated for use in the present invention included herein include, but are not limited to, fluorescent dyes, electron-dense reagents, enzymes (eg, commonly used in ELISA), biotin, digoxigenin, or, for example, , Haptens and proteins that can be made detectable by incorporating a discriminating radioisotope into the peptide or can be used to detect antibodies that specifically react with the peptide. See, eg, US Pat. No. 7,419,779.

In one aspect of the invention, the one or more vectors comprising one or more shRNA of the invention can be readministered at any time interval after the first administration and an unlimited number of times after the first administration.

shRNA: Pharmaceutical Compositions The shRNA-encoding nucleic acids of the invention can be formulated into pharmaceutical compositions and prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, Pa.). The pharmaceutical composition of the present invention comprises a therapeutically effective amount of a vector encoding shRNA. These compositions may contain, in addition to the vector, pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other substances well known to those of ordinary skill in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, eg, intravenous, oral, intramuscular, subcutaneous, intrathecal, neural arch or parenteral administration.

When the vectors of the invention are prepared for administration, they can be combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation or unit dosage form. The total active ingredient in such formulation comprises from 0.1 to 99.9% by weight of the formulation.

In another aspect of the invention, the vector of the invention is suitably formulated by any means that allows a sufficient percentage of the sample to enter the cells to induce gene silencing when it should occur. , Can be introduced into the environment of cells. Many formulations for vectors are known to those of skill in the art and can be used so long as they enter the target cell such that the vector can act.

For example, the vector can be formulated in a buffer such as phosphate buffered saline containing liposomes, micellar structures, and capsids. The pharmaceutical formulation of the vector of the present invention can take the form of an aqueous or anhydrous solution or dispersion, or alternatively an emulsion or suspension. Pharmaceutical formulations of the vectors of this invention can include solubilizing or emulsifying agents, and salts of the type well known in the art as optional ingredients. Non-limiting specific examples of carriers and/or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable saline. Other pharmaceutically acceptable carriers for preparing compositions for administration to an individual include solvents or vehicles such as glycols, glycerols, or injectable organic esters. A pharmaceutically acceptable carrier can contain, for example, a physiologically acceptable compound that acts to stabilize or enhance absorption of the shRNA encoding vector. Other physiologically acceptable carriers include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients, saline. There is water, dextrose solution, fructose solution, ethanol or oil of animal, vegetable or synthetic origin. The carrier may also contain other ingredients, such as preservatives.

It will be appreciated that the choice of pharmaceutically acceptable carrier, including physiologically acceptable compounds, will depend on, for example, the route of administration of the composition. The composition containing the vector can also contain a second reagent, such as a laboratory test agent, a nutritional substance, a toxin, or an additional therapeutic agent. Many agents useful in the treatment of heart disease are known to those of skill in the art and are envisioned for use in combination with the vectors of the present invention.

Formulations of the vector with cationic lipids can be used to facilitate transfection of the vector into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules such as polylysine can be used. Suitable lipids include, for example, Oligofectamine and Lipofectamine (Life Technologies), which can be used according to the manufacturer's instructions.

Appropriate amounts of vector must be introduced and these amounts can be empirically determined using standard methods. Generally, effective concentrations of the individual vector species in the environment of the cells are about 50 nM or less, 10 mM or less, or concentrations of the composition of about 1 nM or less can be used. In other embodiments, the methods utilize concentrations of about 200 pM or less, and concentrations of about 50 pM or less can be used in many situations. One of ordinary skill in the art can determine the effective concentration for any particular mammalian subject using standard methods.

The shRNA is preferably administered in a therapeutically effective amount. The actual amount administered, and rate and time-course of administration, will depend on the condition being treated, the nature and severity of the disease or disorder. Prescription of treatment, e.g. decisions on dose, timing etc., is within the responsibility of general practitioners or specialists, and the disorder, condition or disease to be treated, individual mammalian subject's condition, site of delivery, The mode of administration and other factors known to practitioners are generally considered. Examples of techniques and procedures can be found in Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, Pa.).

Alternatively, targeted therapies can be used to deliver the shRNA-encoding vector more specifically to specific types of cells, through the use of targeting systems such as antibodies or cell-specific ligands. Targeting may be more desirable for a variety of reasons, such as, for example, if the drug is unacceptably toxic, or requires a very high dose, or is unable to enter target cells.

shRNA: Gene Therapy siRNAs can also be delivered to mammalian cells, especially human cells, by gene therapy techniques, for example using DNA vectors in which small hairpin forms (shRNAs) of siRNA compounds can be directly transcribed. Recent studies have shown that double-stranded siRNAs are highly effective at mediating RNAi, but short single-stranded hairpin-shaped RNAs are also likely to be converted to double-stranded siRNAs by intracellular enzymes. It folds into an intramolecular duplex that is processed so that it can mediate RNAi. A cell or tissue is provided with a DNA vector carrying short inverted repeats separated by a few (eg, 3, 4, 5, 6, 7, 8, 9) nucleotides that direct transcription of such a small hairpin RNA. This finding has important and broad implications, as the production of such shRNAs can easily be achieved in vivo by transfecting S. In addition, RNAi produced by the encoded shRNA is stable if a mechanism is included to direct the integration of the vector or vector segment into the host cell genome, or to ensure the stability of the transcribed vector. , And can be inherited. Such techniques have not only been used to “knock down” the expression of specific genes in mammalian cells, but are now also exogenously expressed transgenes, as well as in living mice. It has been used successfully to knock down the expression of endogenous genes in the brain and liver.

Gene therapy is performed according to generally accepted methods as are known to those of skill in the art. See, eg, US Pat. Nos. 5,837,492 and 5,800,998 and references cited therein. In gene therapy, a vector is meant to include a polynucleotide sequence containing sufficient sequences to express the polynucleotide encoded therein. If the polynucleotide encodes an shRNA, expression will produce an antisense polynucleotide sequence. Thus, in this context expression does not require the protein product to be synthesized. In addition to the shRNA encoded by the vector, the vector also contains a promoter functional in eukaryotic cells. The shRNA sequence is under the control of this promoter. Suitable eukaryotic promoters include those described elsewhere herein and are known to those of skill in the art. The expression vector may also include sequences such as selectable markers, reporter genes and other regulatory sequences conventionally used.

Therefore, the amount of shRNA produced in situ depends on the nature of the promoter used to direct transcription of the nucleic acid sequence (ie, whether the promoter is constitutive or regulatable, strong or weak) and intracellular. Are regulated by controlling factors such as the copy number of the nucleic acid sequence encoding the shRNA sequence under.

In the case of Sav1 shRNA expression, the promoter is operably linked to the shRNA sequence. As used herein, the term "promoter" refers to a DNA sequence that regulates the expression of a target gene sequence operably linked to a promoter sequence in a particular host cell. The term "operably linked" means that a nucleic acid fragment is linked to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragments. The expression cassette of the present invention further comprises various expression control sequences, such as an optional operator sequence for controlling transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. be able to. The promoter used in the present invention may be a constitutive promoter that constitutively induces the expression of the target gene, or an inducible promoter that induces the expression of the target gene at a given position and time point. Specific examples of promoters include U6 promoter, CMV (cytomegalovirus) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991; and Monahan et al., Gene Therapy, 7: 24-30, 2000), CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), Rsyn7 promoter (US Patent Application No. 08/991601), ubiquitin promoter (Christensen et al., Plant Mol). Biol. 12:619-632, 1989), ALS promoter (US Patent Application No. 08/409297), and the like. Further, usable promoters include US Pat. Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, It is disclosed in No. 5,399,680, No. 5,268,463, No. 5,608,142 and the like.

The recombinant vector of the present disclosure can be introduced into host cells using conventional methods known to those skilled in the art. Host cells can be utilized for engineering the vector or as a means of transferring the vector to an individual. Preferably, the intracellular integration of the vector into the host cell is by conventional methods known to those skilled in the art such as calcium chloride, biolistic bombardment, electroporation, PEG-mediated fusion, microscopic injection, liposome-mediated methods, and the like. Can be implemented.

Examples of host cells that can be used in the present invention include, but are not limited to, Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis and Staphylococcus. Prokaryotic cells, fungi (eg Aspergillus), yeast (eg Pichia pastoris), yeast (Saccharomyces cerevisiae), fission yeast and lower eukaryotic cells such as Neurospora crassa, and insect cells, plant cells, There may be higher eukaryotic cells such as mammalian cells. Preferably the host cell may be a human cell.

On the other hand, standard recombinant DNA and molecular cloning techniques used in this disclosure are well known to those of skill in the art and can be found in the following literature: Sambrook, J. et al. Fritsch, E.; F. And Maniatis, T.; , Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.M. Y. (1989); Silhavy, T.; J. Bennan, M.; L. And Enquist, L.; W. , Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, N.; Y. (1984); and Greene Publishing Publishing Assoc. and Wiley-Interscience (1987), Ausubel, F.; M. Et al., Current Protocols in Molecular Biology.

The pharmaceutical composition according to the present invention may comprise a therapeutically effective amount of the recombinant vector of the present invention and a cardiovascular drug, alone or in combination with one or more pharmaceutically acceptable carriers. As used herein, the term "therapeutically effective amount" refers to an amount capable of producing more than the desired therapeutic response exhibited by a negative control. Preferably, the therapeutically effective amount is a dose sufficient to prevent or treat cardiovascular disease.

The therapeutically effective amount of the recombinant vector in the present disclosure may range from 0.0001 to 100 mg/day/kg (BW), preferably 0.005 to 0.05 mg/day/kg. However, the effective dose of the drug may vary depending on various factors such as type and severity of disease, age, weight, health and sex of patient, administration route and treatment period.

As used herein, the term "pharmaceutically acceptable" refers to a compound that is physiologically acceptable and causes an allergic reaction (eg, gastrointestinal disorders, and dizziness) or similar reaction when administered to a human or animal. It means that there is no inhibitory effect on the action of the active ingredient. Examples of pharmaceutically acceptable carriers can include any solvent, dispersion medium, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

On the other hand, the pharmaceutical composition of the present invention can be appropriately formulated according to the route of drug administration, by combining it with any appropriate carrier by a conventional method known to those skilled in the art. There is no particular limitation on the route of administration of the pharmaceutical composition. Therefore, the pharmaceutical composition according to the present invention can be administered by the oral or parenteral route. Examples of parenteral routes of administration may include transdermal, intranasal, intraperitoneal, intramuscular, subcutaneous and intravenous routes.

When the pharmaceutical composition of the invention is administered by the oral route, it may be combined with any orally acceptable vehicle according to conventional methods known to those skilled in the art to make powders, granules, tablets, pills. It can be formulated into various dosage forms such as sugar coating, capsules, liquids, gels, syrups, suspensions and cachets. Examples of suitable vehicles include various fillers such as sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol; starches such as corn starch, wheat starch, rice starch and potato starch; cellulose; Cellulose materials such as methyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl methyl cellulose; gelatin, polyvinylpyrrolidone (PVP) and the like. If desired, cross-linked polyvinylpyrrolidone, agar, and a disintegrating agent such as alginic acid or sodium alginate can be added. In addition, the pharmaceutical composition may further comprise anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives.

When the pharmaceutical composition of the invention is administered by the parenteral route, the pharmaceutical composition may be combined with any parenterally acceptable vehicle according to conventional methods known to those skilled in the art, for example, an injectable formulation. , A transdermal preparation or a nasal inhaler. For the preparation of injectable preparations, sterilization must be carried out in combination with protection of the pharmaceutical preparation from microbial contamination, including pathogens and fungi. Examples of suitable vehicles for injectable formulations include, but are not limited to, solvents or dispersions including water, ethanol, polyhydric alcohols (eg glycerol, propylene glycol and liquid polyethylene glycol), mixtures and/or vegetable oils. There can be a medium. More preferably, examples of suitable vehicles include Hank's solution, Ringer's solution, PBS containing triethanolamine (phosphate buffered saline), distilled water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. There may be an isotonic solution. To protect the injectable preparation against microbial contamination, the preparation can further include various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example sugars or sodium chloride.

In the case of a transdermal preparation, the pharmaceutical composition of the present invention can be formulated in the form of an ointment, cream, lotion, gel, external solution, paste, coating or spray. The term "transdermal administration" means that when the pharmaceutical composition is topically applied to the epidermis, a therapeutically effective amount of the active ingredient contained in the pharmaceutical composition is transferred to the epidermis. These formulations are described in the literature which is a well known guide in all pharmaceutical chemistry (Remington's Pharmaceutical Sciences, 15th Edition, 1975, Mack Publishing Company, Easton, Pa.).

For administration by inhalation, the compounds used in the invention may be in a pressurized pack or nebula using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Conveniently delivered in the form of an aerosol spray from Iser. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated to contain a powdered mixture of the compound and a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable vehicles can be found in the literature (Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton, Pa., 1995).

The pharmaceutical compositions of the present invention may comprise one or more buffers (eg saline or PBS), carbohydrates (eg glucose, mannose, sucrose or dextran), antioxidants, bacteriostats, chelating agents (eg EDTA or glutathione). ), adjuvants (eg aluminum hydroxide), suspending agents, thickeners and/or preservatives.

In addition, the pharmaceutical compositions of the present invention may be administered by conventional methods known to those skilled in the art such that the active ingredient may achieve a rapid, sustained or delayed release after administration of the composition to a mammal. It can be appropriately formulated.

Furthermore, the pharmaceutical composition of the present invention can be administered in combination with a known drug having a therapeutic effect for treating a cardiac condition.

The following examples are included to demonstrate certain non-limiting aspects of the invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in the practice of the invention. However, one of ordinary skill in the art will appreciate that similar or similar results may be obtained by one of ordinary skill in the art without departing from the spirit and scope of the invention, many modifications may be made to the particular embodiments disclosed. It should be recognized in light.

Example 1
Hippo signaling promotes adult cardiomyogenesis While other organs have the capacity to regenerate, cardiomyocytes are unable to regenerate or regenerate sufficiently to repair a damaged heart (Kikuchi and Poss, 2012). To examine the hypothesis that Hippo signaling is a negative regulator of postnatal cardiomyogenesis, we inactivated Salv and both Lats genes in adult cardiomyocytes.

To test the role of Salv, Lats1 and Lats2 in adult cardiomyocytes, the conditional null allele was used for the Hippo gene and the Myh6 ^creERT2 transgene that induces tamoxifen-regulated cardiomyocyte cre activity (Sohal et al., 2001. Year). Since the heart contains multiple cell types, to track cardiomyocyte lineages, cardiomyocytes were visualized using the R26 ^mTmG (mTmG) allele, which expresses eGFP in response to cre activation (Muzumdar et al. , 2007). Adult cardiomyocytes that are mutants of Salv and Lats1/2 were generated by injecting tamoxifen into 3-month-old mice (Heallen et al., 2013). To determine if Hippo deficiency resulted in cell cycle resumption, mice were injected with 5-ethynyl-2'-deoxyuridine (EdU). Nuclear EdU integration, which is indicative of de novo DNA synthesis, was detected in both Salv conditional knockout (CKO) and Lats1/2CKO mutant cardiomyocytes, indicating that endogenous cardiomyogenic potential in the absence of Hippo signaling. Is shown. Quantification of EdU-positive cells showed a significant induction of DNA synthesis in Hippo-deficient hearts, with a greater increase in Lats1/2 mutants compared to Salv CKO cardiomyocytes (Heallen et al., 2013). Cell cycle resumption was also quantified in isolated cardiomyocyte nuclei using FACS analysis (Bergmann et al., 2009; Heallen et al., 2013). Both Lats1/2 CKO and Salv CKO cardiomyocyte nuclei increased the number of Ki-67 expressing cardiomyocytes compared to controls (Heallen et al., 2013). These results indicate that cardiomyocytes re-enter the cell cycle in response to the Hippo pathway disruption, supporting the hypothesis that Hippo signaling is a negative regulator of adult cardiomyocyte neogenesis.

It was evaluated whether Salv CKO and Lats1/2 CKO cardiomyocytes develop by mitosis and cytokinesis. Immunohistochemistry was performed with the M phase marker Aurora B kinase (Aurkb) to determine whether cell division occurred in Hippo deficient cardiomyocytes. The apparent detectable Aurkb expression in Lats1/2 and Salv CKO cardiomyocytes in the cleavage furrow provided direct evidence of cell division (Heallen et al., 2013). In contrast to Hippo deficient hearts, Aurkb expression was not detected in control hearts.

In summary, inactivation of the Hippo pathway in the stress-free adult mouse heart results in enhanced cardiomyogenesis with initiation of myocardial S phase and increased mitotic development (Heallen et al., 2013). These findings reveal an inhibitory role for Hippo signaling in adult cardiomyogenesis.

Example 2
Hippo Signaling Promotes Adult Cardiac Regeneration in an Acute Injury Model Apexectomy at the first 6 days of survival leads to cardiac regeneration, whereas resections performed after 7 days of birth (P) are fibrous. It results in formation and scarring (Porrrello et al., 2011).

To test regenerative capacity, uniform sized apex excisions were performed in control and Hippo deficient hearts at the P8 stage, which is not normally regenerated. To inactivate Salv, mice were injected with 4 doses of tamoxifen before and after excision. Both GFP fluorescence detecting recombination in the mTmG reporter and immunofluorescence with anti-Salv antibody showed effective deletion of Salv in mutant myocardium 4 days after excision (4 dpr). Evaluation of 21 dpr hearts by serial excision revealed severe scarring of control hearts in some cases. In contrast, resected Hippo-deficient hearts efficiently regenerated myocardium and reduced scar size (Heallen et al., 2013). The regenerated apex originated mainly from pre-existing cardiomyocytes.

Left anterior descending (LAD) coronary artery occlusion was performed at P8 and 2 months of age. In P8 hearts, analysis of LAD closure (LADO) followed by 21 days after closure showed recovery of function and reduction in scar size. Histological examination also confirmed myocardial recovery and reduced scar tissue after LADO (Heallen et al., 2013). There was similarly strong histological and functional evidence for cardiomyocyte regeneration after LADO in the adult heart. Shortening and ejection fractions assessed by echocardiography showed that by 3 weeks post LADO, adult Hippo deficient hearts had restored equivalent function to that of sham-operated animals. Suggest that Hippo deficient cardiomyocytes have enhanced survival and/or proliferation following ischemic injury (Heallen et al., 2013).

Example 3
HIPPO-deficient cardiomyocytes proliferate extensively during regeneration and acquire migratory properties The 4dpr(P12) Hippo-deficient heart was evaluated in more detail. Four hours before harvest, the heart was pulsed with EdU to visualize cells that entered the cell cycle. In control hearts, EdU-positive cells are found predominantly in GFP-negative, non-cardiomyocyte lineages near excised areas, most likely infiltrating inflammatory cells and proliferative cardiac fibroblasts. Similar proliferative GFP negative cells were also observed in the Salv CKO heart. In contrast to controls, Salv CKO excised hearts had EdU/GFP double-positive cardiomyocytes in both the border zone and the distal heart region (Heallen et al., 2013; Morikawa et al., 2014). ).

Hippo deficiency enhances the ability of cardiomyocytes to re-enter the cell cycle throughout the heart. In the Salv CKO mutant heart, cells from the cardiomyocyte lineage separated from the surrounding border zone cardiomyocytes and entered excised zones containing a large number of non-cardiomyocytes. Furthermore, Hippo-deficient GFP-positive myocardium-derived cells extended lamellipodia-like protrusions. In control hearts, GFP-positive cells clustered within the border zone and did not infiltrate the resected area of the heart (Morikowa et al., 2014).

Example 4
Yap Directly Regulates Genes That Promote Cell Cycle Progression and Cytoskeleton Remodeling Chromatin immunoprecipitation sequencing (ChIP-seq) to gain further insight into the direct targeting of Hippo signaling in cardiac regeneration. Experiments were performed using antibodies to the Hippo effector Yap. The mRNA expression data from the microarray experiments were used to overlay the ChIP-seq dataset with the genes up-regulated in Nkx2.5 ^cre Salv mutant hearts (Fig. 2A, B). Changes in gene expression were confirmed by qRT-PCR experiments (Fig. 2B). Gene ontology analysis showed that Yap directly regulates genes involved in cell cycle progression and cytoskeletal mechanics (FIG. 2A and [Morikawa et al., 2014]). Among the cell cycle genes regulated by Yap are Cdk6 and the previously identified Yap target, cyclin-dependent kinases such as Cyclin E2. Another Yap target, the cell cycle gene Lin9, is a member of the MuvB complex and enhances the G2/M transition (Kleinschmidt et al., 2009; Sadasivam et al., 2012) (FIGS. 2A,B).

Yap target genes that regulate both cytoskeleton and cell cycle progression include Aurkb and Birc5 (survivin) (Fig. 2A, B). Aurkb and Birc5 are components of the chromosomal passenger complex and are important for chromosome condensation and segregation during mitosis and cell division. Importantly, Birc5 was previously shown to be regulated by Hippo signaling in the developing heart (Heallen et al., 2011). Genes expressed in the cytokinesis groove and central spindle that regulate cell division, such as Anillin, Pkp4 and Ect2, are direct Yap target genes to promote cell division when Yap regenerates cardiomyocytes (Hesse et al., 2012; Matthews et al., 2012; Wolf et al., 2006).

Example 5
Yap Directly Regulates Genes That Promote Cytoskeleton Remodeling and Cell Motility During Cardiac Regeneration Consistent with dramatic changes in cell morphology, Yap binds directly to genes that regulate actin cytoskeleton. It was also discovered (Fig. 2A, B). Some of the targets regulated by Yap are known to be localized to lamellipodia and filopodia, such as Enah and Ena/VASP actin regulators that cause cardiac dysfunction when deficient in mice. (Mejillano et al., 2004; Morikawa et al., 2014).

Yap also binds to genes involved in force transmission between cardiomyocytes and the extracellular matrix (ECM). These include genes involved in connecting the actin cytoskeleton to the plasma membrane. Sarcoglycan delta (Sgcd) and syntrophin B1 (Sntb1) are both components of the dystrophin glycoprotein complex (DGC), which is important for linking the actin cytoskeleton to the ECM and between muscle cells. Forces can be transmitted (Barton, 2006; Goyenvalle et al., 2011). Both genes are mutated in human patients with muscular dystrophy and stabilize the plasma membrane in response to mechanical stress. Actin-binding protein (Talin2) links the actin cytoskeleton to integrins and ECM. Finally, Ctna3, a cadherin-binding gene expressed in the intercalated disc (ICD), links the actin cytoskeleton to both ICD and ECM and is likely to detect tension between cardiomyocytes (Li et al., 2012). Year).

Example 6
Hippo ablation rescues abnormal cardiac regeneration in MDX mutant hearts.
To determine whether loss of function of the Hippo pathway could suppress the mdx aplasia phenotype, Salv;mdx double mutant hearts were generated and spleneced at 8 days of age for the double mutant and control samples. Was carried out. As shown in FIG. 3, the Salv CKO and Salv;mdx double mutants regenerated myocardium, in contrast to the mdx mutants that failed to regenerate myocardium (Moriwawa et al., 2014). This indicates that Hippo removal, and thereby upregulation of Yap and upregulation of Yap target gene, can suppress the mdx heart phenotype. This finding indicates that Hippo ablation in the myocardium is a promising approach to treating DMD cardiomyopathy.

Example 7
Hippo Deletion Promotes Cardiomyocyte Regeneration in Established Heart Failure It was determined whether inactivation of the Hippo pathway 3 weeks after myocardial infarction still promotes cardiomyocyte regeneration with improved cardiac function. This is a clinically significant problem as many patients suffer from chronic infarction leading to pathological cardiac remodeling with heart failure and death. To date, it was not known whether Hippo deficiency could effectively promote cardiac regeneration in established mature scars. Myocardial infarction was generated in control and uninjected Salv CKO hearts at time 0 (FIG. 4). Uninjected Salv CKO mice still express control levels of Salv. A sham control was also established at time zero. Three weeks after infarction, all mice were carefully examined by echocardiography and operated mice with heart failure with reduced EF were included in this study, and tamoxifen injection was administered to inactivate the Hippo pathway. did.

Parameters of cardiac function were evaluated in all hearts at 2 week intervals after tamoxifen injection (Fig. 4). The Salv CKO variant began to recover function at 2 weeks (5 weeks after myocardial infarction) and continued to improve until reaching the level of non-operated controls (9 weeks after myocardial infarction). Inactivation of the Hippo pathway after established heart failure results in repair of the heart with restoration of cardiac function.

Example 8
Design of Small Hairpin RNAs (SHRNAs) that Inactivate the Hippo Pathway in the Adult Heart Three examples of shRNAs for Salv are for the conserved region of the Salv molecule between mouse, human and pig, Can be used interchangeably among these three species using standard methods ([Tafer, 2014]; FIGS. 6, 7 and 8). The functional efficiency of each shRNA that suppresses endogenous Salv expression in neonatal cardiomyocytes was confirmed using siRNA knockdown experiments (Fig. 5). All three shRNAs effectively knocked down Salv expression by approximately 70%, with shRNA#3 providing the highest knockdown efficiency.
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While the present invention and its advantages have been described in detail, various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined in the appended claims. Will be understood. Furthermore, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art can readily appreciate from the present disclosure, existing or later developed functions that perform substantially the same function or produce substantially the same results as the corresponding embodiments described herein. Any process, machine, manufacture, composition, means, method or step can be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims (29)

SEQ ID NO 10, SEQ ID NO 11, or, or comprising SEQ ID NO: 12, and / or, SEQ ID NO 10, SEQ ID NO: 11, or at least 95% identity to one of SEQ ID NO: 12 An isolated synthetic nucleic acid composition comprising a derived nucleic acid comprising a sex, wherein the nucleic acid is shRNA, and the nucleic acid is capable of suppressing endogenous Salvador expression in neonatal cardiomyocytes. Separated synthetic nucleic acid composition. The nucleic acid comprises the sequence of SEQ ID NO: 10 and further comprises an antisense sequence of SEQ ID NO: 10, wherein the sequence and the antisense sequence hybridize with each other to form a double-stranded structure, and The composition of claim 1, wherein the antisense sequences are separated by a loop structure. The nucleic acid comprises the sequence of SEQ ID NO:11 and further comprises an antisense sequence of SEQ ID NO:11, and when the sequence and the antisense sequence hybridize with each other to form a double-stranded structure, The composition of claim 1, wherein the antisense sequences are separated by a loop structure. The nucleic acid comprises the sequence of SEQ ID NO: 12, further comprises the antisense sequence of SEQ ID NO: 12, and when the sequence and the antisense sequence hybridize with each other to form a double-stranded structure, The composition of claim 1, wherein the antisense sequences are separated by a loop structure. The composition according to any one of claims 1 to 5 , wherein the nucleic acid is at least 43 nucleotides in length. Wherein the nucleic acid is less than or equal to the length 137 nucleotides, composition according to any one of claims 1 to 5. It said loop structure is a length 5 to 19 nucleotides, composition according to any one of claims 1 to 6. The derived nucleic acid, pre-Sharing, ABS column number 10, having one mismatch compared to the respective SEQ ID NO: 11 or SEQ ID NO: 12, the composition according to any one of claims 1-7. The nucleic acid or derived nucleic acids, are included in the vector, composition of any one of claims 1-8. The composition according to claim 9 , wherein the vector is a viral vector. The composition according to claim 9 , wherein the vector is a non-viral vector. The composition according to claim 9 , wherein the vector is a non-integrating vector. 13. The composition of claim 12 , wherein the non-integrating vector is a lentivirus vector. 10. The composition of claim 9 , wherein expression of the nucleic acid is regulated by a tissue-specific or cell-specific promoter. 15. The composition of claim 14 , wherein the promoter is a cardiomyocyte-specific promoter. The cardiomyocyte-specific promoter is a rat ventricle-specific myocardial myosin light chain 2 (MLC-2v) promoter; myocardium-specific α-myosin heavy chain (MHC) gene promoter; NCX1 promoter -137 to +85 heart cell-specific minimum 16. The composition of claim 15 , wherein the promoter is chicken heart troponin T (cTNT), or a combination thereof. SEQ ID NO 10, two or more of the nucleic acid comprising SEQ ID NO: 11 or SEQ ID NO: 12 are present on the same vector, composition of claim 9. 18. The composition of claim 17 , wherein the two or more nucleic acids are regulated by the same regulatory sequence. 18. The composition of claim 17 , wherein the two or more nucleic acids are regulated by different regulatory sequences. Is for treating heart condition in an individual, the composition according to any one of claims 1 to 19. 21. The composition of claim 20 , wherein the individual requires cardiomyogenesis due to the cardiac condition in the individual. 22. The composition of claim 20 or 21 , wherein the heart of the individual has cardiomyocyte apoptosis, necrosis and/or autophagy. Said cardiac condition, cardiovascular disease, cardiomyopathy, heart failure, myocardial infarction, ischemia, necrosis, fibrosis or diabetic cardiomyopathy, including senile cardiomyopathy, according to any one of claims 20-22 Composition. 21. The composition of claim 20 , wherein the individual has Duchenne Muscular Dystrophy. The composition according to any one of claims 20 to 24 , wherein the composition is given to the individual more than once. The composition according to any one of claims 20 to 25 , wherein the composition is given to the individual systemically. The composition according to any one of claims 20 to 25 , wherein the composition is topically applied to the individual. It said individual is subjected to additional therapy for the cardiac condition, composition according to any one of claims 20-27. A kit comprising the composition of any one of claims 1 to 28 , wherein the composition is held in a suitable container.